An expeditious chemo-enzymatic route from glucose to catechol by theuse of 2-deoxy-scyllo-inosose synthase

Article abstract of DOI:10.1016/S0040-4039(00)00064-2

A potential two-step process to catechol from D-glucose comprising one- pot incubation of D-glucose with recombinant 2-deoxy-scyllo-inosose synthase (BtrC) and hexokinase, togther with chemical reductive dehydration of the resulting 2-deoxy-scyllo-inosose with Hl, was developed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00064-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1935–1938
An expeditious chemo-enzymatic route from glucose to catechol
by the use of 2-deoxy-scyllo-inosose synthase
Katsumi Kakinuma,∗ Eriko Nango, Fumitaka Kudo, Yoshitaka Matsushima and
Tadashi Eguchi †
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
Received 1 December 1999; accepted 28 December 1999
Abstract
A potential two-step process to catechol from D-glucose comprising one-pot incubation of D-glucose with
recombinant 2-deoxy-scyllo-inosose synthase (BtrC) and hexokinase, together with chemical reductive dehydration
of the resulting 2-deoxy-scyllo-inosose with HI, was developed. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: chemo-enzymatic route; catechol; carbocycle forming enzyme; glucose.
Facing the inevitable shortage of fossil resources in the coming centuries, it is highly desirable to
facilitate new technologies for the production of versatile chemicals from sustainable and/or recyclable
resources, preferably using environmentally acceptable methods.1 Frost et al. demonstrated pioneering
works for the production of catechol and other benzenoid chemicals by the use of the enzymes involved
in the shikimate pathway as well as by myo-inositol synthase.2
During our work on the molecular analysis of the biosynthesis of clinically important 2-
deoxystreptamine-containing aminocyclitol antibiotics,3 we were successful in purifying the key enzyme,
2-deoxy-scyllo-inosose synthase (DOIS) from butirosin-producing Bacillus circulans SANK72073.4
Subsequently, its structural gene (btrC) has been identified and heterologously over-expressed recently
in Escherichia coli.5 DOIS catalyzes the multi-step direct cyclization of D-glucose-6-phosphate (G-6-P)
into the six-membered carbocycle 2-deoxy-scyllo-inosose (DOI) as illustrated in Scheme 1.
With over-expressed DOIS in hand, we envisioned that the DOI product may well be conveniently
converted into catechol or other benzenoids by rather simple chemical reactions, because its unique
unsymmetrical structure differs from myo-inositol.2d Catechol is an important industrial chemical used
as an antioxidant, starting material for flavors and fragrances, medical and agricultural agents, and for
other things. This communication concerns preliminary results of a novel chemo-enzymatic two-step
∗
†
Corresponding author. Tel: +81-3-5734-2227; fax: +81-3-5734-3713; e-mail: kakinuma@chem.titech.ac.jp (K. Kakinuma)
Present address: Department of Chemistry and Materials Science, Tokyo Institute of Technology.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00064-2
tetl 16344

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Scheme 1.
approach to catechol from D-glucose. In addition to this potential, DOI itself is an interesting chiral
2,3,4,5-tetrahydroxycyclohexanone resource for asymmetric synthesis.
In order to transform D-glucose to catechol or other benzenoids by way of DOI, two issues had to be
addressed; one was phosphorylation of D-glucose to G-6-P as the substrate of DOIS (BtrC), and the other
was dehydration of DOI to catechol.
As to the first requisite, it seemed logical and appropriate to use hexokinase in the presence of
adenosine triphosphate (ATP) for the convenient preparation of G-6-P, since the enzyme is easily
available and several procedures for the regeneration of the ATP cofactor have been well documented.6
After several attempts, one-pot conversion of D-glucose to DOI turned out to be successful, as shown
in Scheme 2. Thus, simultaneous incubation of D-glucose with commercially available hexokinase and
the recombinant BtrC, the latter being over-expressed in E. coli and purified to homogeneity,5 in the
presence of ATP, nicotinamide adenine dinucleotide (NAD+), and Co++, yielded DOI in up to 38% yield.
It should be pointed out that the yield may well be improved, for example, by the use of a larger amount
of enzymes, since an independent incubation of G-6-P with recombinant BtrC was able to afford DOI
almost quantitatively.7
Scheme 2.
The most crucial was the second issue, since any such reactions, particularly a rather simple reaction,
had not been known. Our efforts were devoted to this problem, and the results are summarized in Table
1.
1,2,4-Trihydroxybenzene was produced as its corresponding acetate under acidic acetolysis or ace-
tylation conditions (runs 1, 2, and 3). Importantly, reductive dehydration (runs 4 and 5) clearly yielded
catechol as a major product. Particularly, treatment of DOI with HI8 allowed direct formation of catechol
in good yield (59%). Thus, catechol can now be produced from DOI in a single chemical step, although
the reaction conditions need to be further optimized.
Typical procedures: One-pot synthesis of DOI from D-glucose by BtrC and hexokinase: A mixture
(total volume 100 µl) of 10 mM D-glucose, purified BtrC (27 µM) and hexokinase (0.2 U/ml, Boehringer
Mannheim) was incubated in the presence of 5 mM NAD+ (Oriental Yeast Co., Tokyo), 10 mM ATP

1937
Table 1
(Tokyo Kasei Kogyo TCI, Tokyo), 5 mM MgCl2, and 0.1 mM CoCl2 at 37°C for 11 h, and additional
incubation was further continued at 46°C for 30 min. The yield was quantitated to be 38% by HPLC after
the reaction product was derivatized to O-(4-nitrobenzyl)oxime.3e
Catechol from DOI: A mixture of 43 mg of 2-deoxy-scyllo-inosose, 4 ml of glacial acetic acid and 2.5
ml of conc. hydroiodic acid was heated under Ar at 70°C for 2 h. To the resulting reaction mixture was
added an aqueous saturated NaHSO3 solution and the whole was extracted four times with 70 ml each
of ethyl acetate. The combined organic extract was dried over anhydrous MgSO4. After being filtered,
the extract was evaporated to dryness to give a residue, which was chromatographed on silica gel (Merck
Kieselgel 60, Art. 7734) using hexane:ethyl acetate (8:1) as solvent to give 17 mg (59% yield) of catechol:
1H NMR (500 MHz, CDCl3) δ 6.87 (m, aromatic), 6.81 (m, aromatic), 5.08 (2×OH, br); 13C NMR (125
MHz, CDCl3) δ 143.5, 121.3, 115.5.
Catechol from G-6-P: A mixture (total volume 20 ml) of 5 mM G-6-P, purified BtrC (0.23 µM) was
incubated in the presence of 0.5 mM NAD+, 50 mM Tris–HCl, 0.2 mM CoCl2 at 46°C for 3 h. The
reaction mixture was evaporated to give a crude DOI (68% yield by HPLC). The residue was subjected
to the reductive dehydration with HI as above to give, after purification by prep. TLC, 2.5 mg of catechol
(15% yield from G-6-P).
The above-mentioned chemo-enzymatic method involves much shorter reaction steps than those
proposed by Frost et al.2b Although further improvement and modification appear to be necessary for
large-scale operations, it should be emphasized here that the present approach has significant potential
for the production of catechol and other benzenoids from widely available D-glucose and further implies
significant opportunity for developing acceptable ‘green’ ways for the production of organochemical
resources from sustainable agricultural and biomass products by utilizing enzymes involved in natural
product biosynthesis.

1938
Acknowledgements
This work was financially supported by the Research for the Future Program of the Japan Society for
the Promotion of Science (JSPS-RFTF96I00302) and a Grant-in-Aid for Scientific Research (11356004)
from the Ministry of Education, Science, Sports and Culture.
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